ES2405323T3 - Melanoma treatment - Google Patents

Abstract

Dexanabinol, or a salt thereof, to use in the treatment of melanoma.

Description

Melanoma Treatment

FIELD OF THE INVENTION

The present invention provides medicaments for the treatment of melanoma. More particularly, the present invention provides dexanabinol, or a salt thereof, for the treatment of melanoma.

BACKGROUND

The incidence of melanoma cases has doubled every year since 1940. Melanoma is currently the sixth most common type of cancer in men, and the seventh most common type of cancer in women. Its incidence is increasing worldwide (Parker, S et al, 1997). The 5-year survival rate for melanoma is 30 to 40%, with malignant melanoma the highest risk of metastatic mortality (Jemal et al, 2001); the spread of the disease to remote organs such as the liver, bones and brain, reduces the 5 years of survival to less than 12%. There is currently no effective long-term treatment for patients with metastatic melanoma (phase IV). Standard chemotherapy guidelines do not provide a significant advantage in terms of the long-term survival of such patients and chemotherapy can be related to a certain degree of morbidity due to toxicity. There is an obvious need to develop new treatments aimed at melanoma, both to prevent the evolution of cancer and to treat advanced disease.

It has been shown that the search for new effective treatments for melanoma is a very complicated challenge. The high resistance to conventional antineoplastic and radiation is a characteristic of melanoma. (Smalley and Eisen, 2003; Strauss et al., 2003). Research has now gone on to identify melanoma genetic mutations and corresponding alterations of signal transduction pathways, with the hope that more specific targeted treatments can be developed. It has been shown that various cell pathways important for cell multiplication, apoptosis and resistance or metastasis are activated in melanoma. Melanoma tends to develop a plurality of abnormalities, including the loss of regulatory functions or the acquisition of antiapoptotic or proliferative functions. Therefore, therapeutic agents that can inhibit several signaling pathways simultaneously are intended. In addition, the administration of standard antineoplastic agents together with new agents can give traditional antineoplastic chemotherapies a new boost in the context of melanoma, if resistance to antineoplastic chemotherapy is inhibited. Currently the challenge includes developing selective agents that address these aberrant pathways.

Aberrant activation of the kappa B nuclear factor pathway (NFκB) has been linked to melanoma growth, metastasis and apoptosis escape. In vitro studies have demonstrated the constitutively elevated NFκB binding in human melanoma cultures compared to normal melanocytes (Dhawan et al, 2004, McNulty et al, 2004). It has also been shown that the expression of the NFκB, RelA subunit is significantly elevated in human nevi and melanoma compared to normal skin. The NFκB pathway is therefore a potential target for the treatment of melanoma. The constitutive activation of NFκB has several effects. Previous studies showed that persistent activation of NFκB caused the increase

of the production of chemokines (for example, CXCL8 and CXCL1) that stimulate angiogenesis, stimulating this

Mode of tumor formation in melanoma. In addition, activated NFκB is an important central regulator of a

a certain number of genes involved in antiapoptosis and reproduction (Mayo et al, 1997). Apart from its function

antiapoptotic, NFκB can also play a critical role in the development of resistance to

Antineoplastic chemotherapy in melanoma (Wang et al, 1999).

It has been shown that the activation of NFκB in melanoma is due to the constitutive activation of IKK, a key regulator of the NFκB pathway (Yang and Richmond, 2001). NFκB pathway inhibitors and, in particular,

IKK inhibitors can be a very effective means to kill tumor cells by orienting them to apoptosis.

A small molecule that selectively directs the activation of NFκB in melanoma without affecting the functions of NFκB in normal cells, therefore has a significant therapeutic potential, as a single agent or together with existing antineoplastic chemotherapies. US Patent No. 2006/0025419 discloses the use of an NFκB inhibitor (BMS-345541) for the treatment of melanoma.

A particular compound of interest is 1,1-dimethylheptyl- (3S, 4S) -7-hydroxy-Δ6-tetrahydrocannabinol (dexanabinol) disclosed in US Patent No. 4,876,276.

Apart from being a non-competitive NMDA receptor blocker, dexanabinol has been shown to inhibit NFκB (Juttler et al, 2004).

SUMMARY OF THE INVENTION

A method for rapidly killing melanoma cells comprising contacting the cell with 1,1-dimethylheptyl- (3S, 4S) -7-hydroxy-Δ6-tetrahydrocannabinol (INN dexanabinol), or a salt thereof has been discovered. . The present invention considers melanoma cancer cells that are precancerous, malignant, metastatic or multiresistant.

Therefore, according to a first aspect, the present invention provides dexanabinol, or a salt thereof, for the treatment of melanoma.

The treatment of melanoma according to the present invention may comprise inhibition of NFκB activity in a melanoma cancer cell, providing dexanabinol, or a salt thereof, to the cell.

Alternatively, the treatment of melanoma may comprise the inhibition of oncogenesis of a melanoma cancer cell by contacting the cell with an effective amount of dexanabinol, or a salt thereof. Incogenesis inhibition comprises causing both cytotoxicity and apoptosis in the cancer cell.

In addition, the treatment of melanoma according to the present invention may independently, simultaneously or sequentially comprise inhibition of NFκB activity and oncogenesis of a melanoma cancer cell.

Dexanabinol, or a salt thereof, is advantageous for the treatment of melanoma, among other aspects, because it has a reduced toxicity, less side effects and / or a decrease in resistance compared to the currently used antineoplastic drugs.

It is further considered that a second therapeutic agent together with dexanabinol, or a salt thereof, can be provided to a melanoma cancer cell for the treatment and / or prevention of melanoma. The second agent may comprise an antineoplastic, an immunotherapeutic, a genotherapy or radiotherapy agent. The second therapeutic agent can be administered with dexanabinol, or a salt thereof, independently, simultaneously or sequentially.

Suitable dexanabinol salts are well known and are described in the prior art. Salts of organic and inorganic acids and bases can be used to make pharmaceutically acceptable salts. Said acids comprise, without limitation, hydrofluoric, hydrochloric, hydrobromic, iohydric, sulfuric, nitric, phosphoric, citric, succinic, maleic and palmitic acids. The bases comprise compounds such as sodium and ammonium hydroxides. Those skilled in the art are familiar with quaternization agents that can be used to make pharmaceutically acceptable quaternary ammonium derivatives of dexanabinol. These comprise, without limitation, methyl and ethyl iodides and sulfates.

Accordingly, and in one embodiment, the present invention provides a use of dexanabinol, or a salt thereof, in the manufacture of a medicament for the treatment of melanoma.

In particular, a use of dexanabinol, or a salt thereof, is provided in the realization of a medicament that can be administered topically, for example, a medicament that can be administered topically for the treatment of melanoma.

In addition, in a second aspect, the present invention provides a compound for use in a melanoma treatment method, said method comprising administering a therapeutically effective amount of dexanabinol and salts and / or combinations thereof.

Dexanabinol and salts and / or combinations thereof are known per se and can be prepared using methods known to those skilled in the art or can be obtained commercially. In particular, dexanabinol and the processes for preparing it are described in US Patent No. 4,876,276.

Advantageously, the compound and the salts and / or combinations thereof can be administered orally or intravenously.

Therefore, the compound can be presented as a tablet, capsule, dragee, suppository, suspension, solution, injection, for example intravenously, intramuscularly or intraperitoneally, implant, topical, for example transdermal, preparation such as a gel, cream, ointment, aerosol or a polymer system, or a form for inhalation, for example, an aerosol or a powder formulation.

Compositions suitable for oral administration comprise tablets, capsules, dragees, liquid suspensions, solutions and syrups;

Compositions suitable for topical administration to the skin comprise creams, for example oil-in-water emulsions, water-in-oil emulsions, ointments, gels, lotions, ointments, emollients, colloidal dispersions, suspensions, emulsions, oils, aerosols, foams, mousses and the like. Compositions suitable for topical application may also comprise, for example, liposome vehicles composed of lipids

or special detergents. Examples of other additives, diluents or vehicles are:

for tablets and dragees - substances with a filler function, for example, lactose, starch, cellulose microcrystalline, talc and stearic acid; lubricants / glidants, for example, magnesium stearate and carbon dioxide colloidal silicon; disintegrants, for example, sodium starch glycolate and sodium carmellose; for capsules - pregelatinized starch or lactose; for oral or injectable solutions or enemas - water, glycols, alcohols, glycerin, vegetable oils; for suppositories - natural or hardened oils or waxes.

It may be possible to administer the compound or salts and / or combination thereof or any dosage schedule. combined as described above, transdermally, for example, with a device for transdermal administration or a suitable vehicle or, for example, in an ointment base, which can be incorporated in a patch for controlled administration. Such devices are advantageous, since they can allow a prolonged period of treatment with regard to, for example, an oral or intravenous medication. Examples of transdermal delivery devices may comprise, for example, a patch, dressing, bandage or plaster capable of releasing a compound or substance through the skin of a patient. An expert in subject will be familiar with the materials and techniques that can be used for administration transdermal of a compound or substance and examples of delivery devices are provided transdermal in documents GB2185187, US3249109, US3598122, US4144317, US4262003 and US4307717.

For the treatment of melanoma, the present invention especially allows the administration of compositions topically in the skin or by injection, for example, subcutaneously, or both. In addition, dexanabinol, or a Salt thereof, can be administered simultaneously or sequentially by an injection procedure and by topical route to reduce the growth of a skin tumor.

Topical use compositions are particularly preferred.

The invention will now be illustrated by way of example only.

DETAILED DESCRIPTION

Example 1

Induction of dexanabinol apoptosis in human melanoma cell lines

Methods

3 Human melanoma cell lines (A375, G-361, WM266-4) were maintained in RPMI 1640 culture medium (Sigma, UK) containing 10% (v / v) thermo-activated bovine fetal serum (Sigma, UK) and 2 mM of L-glutamate at 37 ° C in humidified CO2 at 5%.

The cells were collected, washed, and resuspended in culture medium and counted (Beckman-Coulter Vi-CELL XR). The cells were seeded in 240 wells of 384 tissue culture plates in 1.6x105 to 2.4x105 cells / ml in samples of 12.5 μl / well. Samples of 50 µl of culture medium were prepared in the outer wells. 2 plates were prepared for each cell line. The plates were incubated overnight at 37 ° C, in humidified CO2 at 5%.

Dexanabinol was prepared in culture medium at 2 times the final test concentration at 125, 31.3, 7.81, 2.00, 0.49,

0.12, 0.031 and 0.008 μM (the concentration of dimethyl sulfoxide (DMSO) remained constant throughout the entire

0.5% dilution range).

Cisplatin was used as a positive control. The concentrations of the final trials were 10, 2.5, 0.63, 0.156,

0.039, 0.010, 0.002 and 0.0006 μg / ml. 12.5 μl per well of dexanabinol or cisplatin dilutions were added to the plates in replicas of 6. 12.5 μl of culture medium was added to the media of the control wells. The plates were incubated for 24 hours at 37 ° C, in humidified CO2 at 5%.

Caspase levels 3/7 were analyzed using the Apo-ONE © Homogeneous Caspase-3/7 test kit. Fluorescence was determined using a FlexStation® II384 plate reader after 1, 2, 3 and 4 hours after the addition of the caspase substrate. The 4-hour readings were used in the analysis.

The cell viability test was performed in parallel on the same plate for each line using the CellTiter-Blue® reagent (Promega). Simply put, 25 µl of the CellTiter-Blue® reagent (Promega) was added to each well. The plates were stirred for 1 minute at 500 rpm and incubated at 37 ° C, with 5% CO2 for 4 hours. Fluorescence was determined using a FlexStation © II384 plate reader (570 nm wavelength of

5 excitation, 600 nm emission wavelength, 590 nm cutoff value). Graphical representations that show the cytotoxic effect of dexanabinol and cisplatin are shown superimposed on the same graph.

Results

10 The induction of apoptosis in melanoma A375, G-361 and WM266-4 cell lines after 24 hours of incubation with cisplatin or dexanabinol is shown in Figures 1 to 3, respectively, and is summarized in Table 1. It is shown in Table 1. also the analysis of cell viability determined by the CellTiter-Blue® test that indicates cytotoxicity.

15 Cisplatin was used as a positive control and a cytotoxic response was observed in the 3 melanoma cell lines with an approximate IC50 value of 20 to 60 µM. The induction of apoptosis was not so easy to quantify due to inadequate dosage curves (G-361, WM266-4).

Dexanabinol causes a cytotoxic response with IC50 values between 10 and 21 μM at 3

20 melanoma cell lines. The induction of apoptosis was not quantified in G-631 and A375 due to inadequate dose-response curves. A maximum response in apoptosis occurred at 2.5 μM and decreased to the highest concentration of 10 μM possibly due to lysis and cell loss.

Table 1: Effect of dexanabinol on the induction of apoptosis and cell multiplication in 3 cell lines of human melanoma.

Cell line

Cisplatin Dexanabinol

↑ Apoptosis EC50 (µM)

↓ IC50 viability (µM) ↑ Apoptosis (µM) EC50 ↓ IC50 viability (µM)

A375

5.67 ** 21.8 ** ND *** 19.16 **

G-361

Approx. 33.3-100 ** 18.00 ** ND * 10.97 ***

WM266-4

Approx. 33.3-100 *** 62.00 * 13.04 *** 20.87 **

ND - EC / IC50 not determined due to a dose curve - inadequate response Position * weak induction of apoptosis and decreased multiplication (<35%) ** moderate induction of apoptosis and decreased multiplication (35-70% ) *** good induction of apoptosis and decreased multiplication (> 70%)

The results are illustrated in Figures 1 to 3, in which;

Figure 1 illustrates the effects of cisplatin (A) and dexanabinol (B) on apoptosis and the growth of melanoma A375 cells; Figure 2 illustrates the effects of cisplatin (A) and dexanabinol (B) on apoptosis and the growth of melanoma G361 cells; Y

Figure 3 illustrates the effects of cisplatin (A) and dexanabinol (B) on apoptosis and the growth of melanoma WM366-4 cells;

Example 2

40 Inhibition of the multiplication of melanoma cells by dexanabinol

Methods

The ability of dexanabinol to inhibit the multiplication of melanoma cells in 3 45 melanoma cell lines (A375, UACC62, Malme-3M) using the sulphordamine B (SRB) assay was also studied.

Sterile solutions of the final concentrations of dexanabinol (0.001 μM to 100 μM in 0.5% of

DMSO).

50 Cells were incubated with drug concentrations in 0.5% DMSO (100 µl total volume per well) at 37 ° C in 5% CO2 for 5 days. The control wells contained cells plus 5% DMSO plus medium.

The SRB multiplication inhibition assay was performed for 24 days and 5 periods of exposure. After exposure, the cells were fixed, stained with SRB and the reading was done on a SpectorMax® 250 microplate spectrophotometer system plate reader.

Results

The effect of dexanabinol on the inhibition of multiplication in 3 melanoma cell lines after 5 days of exposure is shown in Figure 4. Growth inhibition was also determined after 24 hours of exposure. The IM50 values for both exposure times are summarized in Table 2.

Table 2: Effect of exposure time on the ability of dexanabinol to inhibit the multiplication of melanoma cells in vitro.

A375

Malme-3M UACC62

IM50 (µM) to

5 days 16.2 13.3 18.5

14.4

13.3 18.1

24 hours

14.3

a Determined by the SRB multiplication inhibition assay

Example 3

Effect of dexanabinol on the temporal evolution of cell multiplication

15 Methods

The effect of the temporal evolution of dexanabinol on cell multiplication in a melanoma cell line, the A375, was examined using the clonogenic assay procedure.

A375 cells were collected from a multiplication cell culture and counted. The cells were diluted to 1.0x105 / ml and 2 ml / well seeded in 6-well plates.

The cells were incubated overnight at 37 ° C, in a humidified incubator with 95% air + 5% CO2.

25 Cells were treated with dexanabinol in 4 doses of up to 20 μM in 3 exposure periods (1 hour, 6 hours and 24 hours).

After each exposure period, the cells were collected from the wells by washing twice with PBS and then adding 0.2 ml of a single dose of trypsin to each well and incubating at 37 ° C until the cells separated. Cell suspensions were counted in a dilution of 1/10 and then diluted to 1/10, 1/100, 1/1000. 10 cm plates (containing 7 ml of fresh medium) were planted with 3 different cell densities for dexanabinol treatment and control treatment. When colonies with an adequate size were formed in the control plates, the plates were fixed, stained and counted.

35 The survival percentage of A375 cells treated with dexanabinol was calculated using the following equation:

% Cloning efficiency = Colonies counted X 100 Cultured cells

% Survival = Efficiency of cloning in cells treated with the drug X 100 Efficiency of cloning in control cells

Results

The effect of the temporal evolution of dexanabinol on cell death in melanoma A375 cells is shown in Figure 5.

Example 4

Inhibition of the multiplication of the cells of a human melanoma xenograft by dexanabinol

Methods:

Once it has been determined that dexanabinol inhibits the multiplication of melanoma cells in vitro, it was then attempted to establish whether the compound was active in vivo. A preliminary pharmacokinetic (PK) and maximum tolerated dose (MTD) study was conducted to determine if therapeutically effective dose levels of dexanabinol CD1 mice with A375 tumors could be achieved. The results showed that with the MTD (single iv dose of 100 mg / kg) a plasma concentration of 10 μM was achieved for 2 hours. Based on this, a study of the efficacy of a single dose was performed.

Dexanabinol was diluted in a 10% cremofor / ethanol vehicle (1: 1 v / v) in saline. Control mice were administered the vehicle alone.

100 mice / kg of dexanabinol were administered intravenously to 10 mice. The vehicle (10 ml / kg) was administered to 10 mice. 1 x 107 human melanoma A375 cells were implanted in the mice in 50 µl of medium on the side. Once the tumors were palpable (approximately 5 mm x 5 mm), 20 mice were treated with dexanabinol or with the vehicle (as described above). Mice were observed at least once a day and weighed 3 times a week. Tumor size was determined 3 times a week after treatment. The treatment lasted 4 weeks.

Results

The temporal evolution of tumor growth and a graph that summarizes the evolution until the tumors reach a tumor volume 4 times higher than the day of treatment (period up to RTV 4) are shown in Figures 6 and

7.

SUMMARY

Dexanabinol was analyzed against various established cell lines of human melanoma, including some obtained from metastases of disseminated melanoma in other tissues. In vitro proliferation assays of the cells demonstrated that dexanabinol is very cytotoxic for all human melanoma cell lines studied at concentrations distributed around an average of 14 µM. Dexanabinol caused apoptotic cell death in a way that depended on caspase 3/7.

This effect was independent of time, and cell death was of the same magnitude after 1 hour than after 24 hours.

The in vitro antitumor effects of dexanabinol were observed at drug concentrations that had been shown to be safe and clinically achievable in patients (~ 10-20 μM). The effect of a single dose of dexanabinol on the growth of human melanoma xenografts of cells was studied below. The minimum required plasma concentration, based on the results of the in vitro cell multiplication test, (10 μM was maintained for at least 2 hours) was achieved by administering a single iv dose of dexanabinol to the MTD (100 mg / kg) in hairless mice CD1. The single dose of dexanabinol caused a delay in multiplication effect in CD1 hairless mice bearing the human tumor xenograft A375.

Claims (15)

one.

Dexanabinol, or a salt thereof, to use in the treatment of melanoma.

2.

 Dexanabinol for use according to claim 1, wherein the melanoma cancer cells are precancerous, malignant, metastatic or multi-resistant.

3.

 Dexanabinol for use according to claim 1, wherein the melanoma treatment comprises

independently, simultaneously or sequentially the inhibition of NFκB activity of a cancer cell of melanoma.

Four.

 Dexanabinol for use according to claim 1, wherein the treatment of melanoma comprises the inhibition of oncogenesis of a melanoma cancer cell.

5.

 Dexanabinol for use according to claim 4, wherein the inhibition of oncogenesis comprises causing both cytotoxicity and apoptosis in the cancer cell.

6.

 Dexanabinol for use according to claim 1, together with a second therapeutic agent selected from one or more of an antineoplastic, an immunotherapeutic, a genotherapy or radiotherapy agent.

7.

 Use of dexanabinol in the realization of a medication for the treatment and relief of melanoma.

8.

 Use according to claim 7, wherein the melanoma cancer cells are precancerous, malignant, metastatic or multi-resistant.

9.

 Use according to claim 7, wherein the melanoma treatment comprises independent, simultaneous or

sequentially the inhibition of NFκB activity of a melanoma cancer cell.

10.

 Use according to claim 7, wherein the treatment of melanoma comprises the inhibition of oncogenesis of a melanoma cancer cell.

eleven.

 Use according to claim 10, wherein the inhibition of oncogenesis comprises causing both cytotoxicity and apoptosis in the cancer cell.

12.

 Use according to claim 7, that in carrying out a polytherapy comprising dexanabinol together with one or more therapeutic agent (s) comprising (a) an antineoplastic, an immunotherapeutic, a gene therapy or radiotherapy agent; independently, simultaneously or sequentially.

13.

 Use according to claim 7, wherein the medicament is for oral or intravenous administration.

14.

 Pharmaceutical composition comprising dexanabinol, or a salt thereof, and one or more of a therapeutic agent comprising an antineoplastic, an immunotherapeutic, a gene therapy agent or a radiotherapy agent; in admixture with a pharmaceutically acceptable additive, diluent or carrier for use in the treatment or relief of melanoma.

fifteen.

 Pharmaceutical composition according to claim 14, for use according to claim 14 suitable for topical or intravenous administration.

Figure 1: Effects of cisplatin (A) and dexanabinol (B) on apoptosis and the growth of melanoma A375 cells;

A. Cisplatin

B. Dexanabinol

Caspasa 3/7

Caspasa 3/7

Caspasa 3/7 - RFU

CellTiter-Blue CellTiter-Blue - RFU  Caspasa 3/7 - RFU CellTiter-Blue CellTiter-Blue - RFU

Log concentration (g / ml)

Log concentration (M)

Figure 2: Effects of cisplatin (A) and dexanabinol (B) on apoptosis and the growth of melanoma G-361 cells

A. Cisplatin

B. Dexanabinol

Caspasa 3/7

Caspasa 3/7

Caspasa 3/7 - RFU

CellTiter-Blue CellTiter-Blue - RFU  Caspasa 3/7 - RFU CellTiter-Blue CellTiter-Blue - RFU

Log concentration (g / ml)

Log concentration (M)

Figure 3: Effects of cisplatin (A) and dexanabinol (B) on apoptosis and the growth of melanoma WM366-4 cells

A. Cisplatin

B. Dexanabinol

Caspasa 3/7

Caspasa 3/7

Caspasa 3/7 - RFU

CellTiter-Blue CellTiter-Blue - RFU  Caspasa 3/7 - RFU CellTiter-Blue CellTiter-Blue - RFU

Log concentration (g / ml)

Log concentration (M)

Figure 4: Effect of dexanabinol (B) on the multiplication of cell lines A375 (A), UACC62 (B) and Malme3M (C) of melanoma (5-day exposure) Figure 5: Effect of dexanabinol on the multiplication of melanoma A375 cells after 1 hour, 6 hours and 24 hours of exposure) Figure 6: Tumor evolution in CD1 hairless mice with A375 treated with dexanabinol Day Figure 7: Graph summarizing the period up to RTV 4 in hairless mice CD1 with A375  REFERENCES CITED IN THE DESCRIPTION This list of references cited by the applicant is presented solely for the convenience of the reader. It is not part of the European patent document. Although the collection of references has been done very carefully, errors or omissions cannot be ruled out and the European Patent Office declines all responsibility in this regard. Patent documents cited in the description